POS0411 PROFILING THE HISTONE LANDSCAPE IN OSTEOARTHRITIS: ENRICHMENT OF HISTONE VARIANT H3.3 AND ITS ASSOCIATED POST-TRANSLATIONAL MODIFICATIONS
Authors: C. Núñez Carro, V. Calamia, P. Fernández Puente, M. Blanco-Blanco, P. Quaranta Díaz, T. Hermida Gómez, C. Ruiz-Romero, F. J. Blanco, M. C. De Andres
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Nucleosomes, the basic structural unit of chromatin, are composed of DNA wrapped around an octameric core of histone proteins. The latter is usually comprised by canonical histones H2A, H2B, H3 and H4<sup>[1]</sup>. These can be replaced by histone variants, which provide structural and functional changes to the nucleosome<sup>[2]</sup>. Histones are prone to undergo post-translational modifications (PTMs) that can modify chromatin conformation to induce changes in gene expression<sup>[3]</sup>. Recent evidence supports that epigenetic mechanisms, such as DNA methylation and non-coding RNA modifications, are involved in the pathogenesis of osteoarthritis (OA). Nevertheless, histone modifications mainly remain an unexplored epigenetic mechanism in OA. <h3>Objectives</h3> The aim of the present study was to identify, characterize and compare the histone profile in human articular cartilage samples from normal (N) and OA patients to provide novel insights into the role of histones in the development of OA. <h3>Methods</h3> Histones were extracted from 3 N and 3 OA cartilage samples following a protocol based on acid extraction previously described by our group. Prior to mass spectrometry (MS) analysis, samples were reduced with dithiothreitol and alkylated with iodoacetamide. After an overnight digestion with trypsin, peptides were desalted via StageTips. Samples were injected on a nanoElute LC coupled to a high-resolution timsTOF (Bruker). Peptides were analyzed in DDA mode with PASEF enabled. Raw data were processed using PEAKS Studio 10.6. software against the Human UniProtKB/Swiss-Prot database. Search parameters were: trypsin digestion, one missed cleavage allowed, 15.0 ppm parent mass tolerance, 0.05 Da fragment mass tolerance and carbamidomethylation as fixed modification. To identify and quantify the most studied biological modifications, "PTM" and "Label-free" modules from PEAKS were employed. <h3>Results</h3> Our histone extraction protocol successfully enriched the samples for subsequent MS analysis. The most detected PTMs are among the most studied, including methylation, acetylation, ubiquitination, phosphorylation, deamidation, biotinylation, citrullination, and ADP-ribosylation. PTMs were found in core histones H2A, H2B, H3, H4 and linker histone H1. Moreover, PTMs were detected in some of their variants. Strikingly, we found that the histone variant H3.3 is significantly increased in OA cartilage compared to N cartilage (2.67×104 ± 9.56×103 vs. 3.11×103 ± 1.71×103) (Figure 1 A). Further H3.3 analysis showed three differentially abundant PTMs between OA and N (Figure 1 B). Acetylation of lysine 18 (K18ac) was more abundant in N cartilage. Monomethylation of K79 (K79me1) was upregulated in OA samples. Likewise, dimethylation of K36 (K36me2) was also more abundant in OA cartilage. <h3>Conclusion</h3> The histone variant H3.3, which associates with decondensed states of chromatin and transcriptionally active sites, is upregulated in human OA cartilage. Furthermore, we identified three H3.3-associated PTMs that are differentially expressed in OA and N cartilage. These data provide new insights into the epigenetic landscape of OA and could contribute to the search for novel therapeutic targets to treat the disease. <h3>References</h3> [1]Kouzarides T. Chromatin Modifications and Their Function. <i>Cell</i>. 2007;128(4):693-705. doi:10.1016/j.cell.2007.02.005 [2]Singh R, Bassett E, Chakravarti A, Parthun MR. Replication-dependent histone isoforms: a new source of complexity in chromatin structure and function. <i>Nucleic Acids Research</i>. 2018;46(17):8665-8678. doi:10.1093/nar/gky768 [3]Millán-Zambrano G, Burton A, Bannister AJ, Schneider R. Histone post-translational modifications — cause and consequence of genome function. <i>Nat Rev Genet</i>. 2022;23(9):563-580. doi:10.1038/s41576-022-00468-7 <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.